Background: Delays in accurate diagnosis of drug resistant tuberculosis (DR-TB) can hinder treatment. Whole genome sequencing (WGS) provides more information than standard molecular and phenotypic testing, but commonly used platforms are expensive to implement, and data interpretation requires significant expertise. Aims: We aimed to optimise a TB WGS diagnostic pipeline balancing user-friendliness, cost-effectiveness and time to results, whilst ensuring accuracy. Materials and methods: Growth conditions, DNA extraction protocols and Oxford Nanopore Technologies (ONT) library preparation kits were compared. Software for basecalling and analysis were evaluated to find the most accurate resistance SNP and lineage predictor. Results: Optimally, a spin-column CTAB DNA extraction method was combined with the RBK110.96 library preparation kit, high accuracy basecalling and data analysis using TB-Profiler. Compared with Illumina, the pipeline was concordant for 16/17 (94%) isolates (lineage) and for 17/17 (100%) isolates (resistance SNPs). Our pipeline was 71% (12/17) concordant with phenotypic drug susceptibility test (DST) results. Time-to-diagnosis was around four weeks. Conclusions: This optimised TB sequencing pipeline requires less time expertise to run and analyse than Illumina, takes less time than phenotypic DSTs and the results are comparable with Illumina. The cost per sample is comparable with other methods. These features make it an important tool for incorporating into routine DR-TB diagnostic pipelines and larger scale drug resistance surveillance in all settings.

This project obtained technical support from Oxford Nanopore Technologies Ltd (ONT), but ONT did not contribute financially to the study

https://pandora.tghn.org/sequencing/tuberculosis-sequencing-pipeline/

This work was supported by the regional network of excellence CANTAM (Central Africa Network on Tuberculosis, HIV/AIDs and Malaria), funded by European and Developing Countries Clinical Trials Partnership (EDCTP). Awarded to AZ and FN. https://cantam.net/en/. The study sponsors had no role in the design, data collection, analysis or write up of this study. No funding (real or in kind) was received from ONT.

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All genetic fasta data obtained in this study is available under the Project accession number PRJEB68143 on the European Nucleotide Archive upon publication. The full manual, including further information and tutorial videos, relating to this protocol is available on the PANDORA-ID-NET Global Health Network hub pages: https://pandora.tghn.org/sequencing/tuberculosis-sequencing-pipeline/. All data and additional material is Open Access.